Project Summary Natural products make up the majority of current therapeutic compounds, with many of the over 200,000 known compounds currently untested. Plant natural products are typically in low abundance, difficult to extract from their natural source, and nearly impossible to synthesize with organic chemistry. The use of synthetic biology holds great promise for producing significant quantities of these complex compounds, including alkaloids, flavonoids, and terpenoids. Access to over 6,000 flavonoids results from the stepwise modification of these polyketides by an incredibly diverse set of enzymes including the family of cytochrome P450 enzymes (P450s), and glycotransferases; however, a major limitation to the design-build-test cycle for engineering microbes is that heterologous proteins often fail to properly fold in a non-native host. It is clear that solving the common modes of failure of heterologous protein expression will reduce strain engineering effort, enable more successful screening of natural product pathway libraries, and greatly increases the number of functional natural enzyme variants. In this MIRA project, we are undertaking the following three projects to demonstrate the use of cellular stress response to debug plant natural product libraries: 1) Detecting common modes of failure of heterologous protein expression; 2) Engineering strains of yeast that identify and fix protein expression failure modes; 3) Improving flavonoid and monoterpene indole alkaloid biosynthesis through improved enzyme expression and metabolic engineering. We are building on our strength engineering Yarrowia lipolytica as an alternative to S. cerevisiae that has naturally higher flux towards key intermediates for plant natural product biosynthesis, such as acetyl-CoA, malonyl-CoA, E4P and NADPH. In all three of these projects, we are seeking to increase the throughput of strain design. The work focuses primarily on improving strain design through genetic implementation; however, our ability to measure the effectiveness of the strain designs is currently limited by the throughput of our analytical methods. This supplement requests the purchase of a GC-MS that seeks to (i) establish higher-throughput capability to analyze flavonoids, terpenes and alkaloids, and variations thereof while (ii) seeding new multi-disciplinary collaboration within Clemson University.